The rapid detection and identification of medically important organisms and macromolecular entities such as bacteria, viruses, malignant cells and the like is of critical importance in establishing diagnoses, treating patients, tracing the source of infections, detecting biological contaminations, and routinely screening and monitoring blood, tissue banks and food so that public health might not be compromised.
The ability to detect and identify pathogenic organisms and macromolecular entities is limited by the sensitivity and rapidity of the detection system. In addition, identification of pathogenic organisms in blood or tissue samples poses special problems; not only is the availability of sample limited but the concentration of the pathogenic organism in that sample is often very low.
Techniques based on molecular methods of detection such as nucleic acid hybridization, restriction enzyme analysis, Southern analysis, Northern analysis, Western analysis and immunoassay have not overcome the problem of detecting low levels of pathogenic entities in dilute conditions. In many cases it is necessary to first incubate samples suspected of containing a pathogenic organism so as to enrich and increase the number of organisms to identifiable levels before detection and identification is possible. Andrews, W.H., Food Tech. 39:77-82 (1985). However, growth and enrichment steps are extremely time-consuming in situations where time is of the essence in establishing the presence or identity of an infectious organism.
In addition, growth requirements for some organisms are very complex and false negatives are a concern. Lack of growth of a bacterium may only indicate that the growth conditions weren't favorable, or that other, nonpathogenic bacteria in the sample grew faster than the organism in question and successfully "competed it out".
Methods for the detection and identification of pathogenic entities such as viruses are even more complex then those for entities like bacteria. More commonly, they depend on the acute and convalescent measurement of a serologic or antibody response to the infectious agent. These measurements are often time-consuming. They often depend on identification and use of a suitable cell line which the virus can infect and in which the virus can replicate. They may also depend on the identification of an animal host which the virus can infect and in which the virus will induce diagnostic serological symptoms.
Thus the identification of a pathogenic organism in blood and tissue samples may be missed even though the organism was present in the sample at levels infectious to humans.
Specific affinity reagents such as high affinity monoclonal antibodies have, in some cases, made it possible to confirm the presence, in blood or tissue samples, of organisms known or suspected of being infectious or otherwise pathogenic. For example, high affinity monoclonal antibodies directed to the hepatitis B surface antigen (HB.sub.s Ag) have been developed, Wands, J.R., et al., Gastroenterology 80:225-232 (1981). These antibodies have successfully identified the presence of hepatitis B virus or its variants in low levels in the blood and tissues of some patients with acute and chronic liver disease but with no known serologic marker of recent or past hepatitis B infection and also in some "healthy" individuals who had no clinical symptoms, Ben-Porath, E. et al., Progress in Liver Diseases 8:347-366 (1986); Ben-Porath, E. et al., J. Clin. Invest. 76:1338-1347 (1985).
However, studies using a monoclonal antibody have been limited because it has been impossible to further characterize or study the molecular identity of the virus or variant in these patients. Levels of the virus, although detectable with the monoclonal antibody, are often too low for cloning, sequencing and other methods of viral characterization. See, e.g., Dienstag, J.L. et al., in Harrison's Principles of Internal Medicine. R.G. Petersdorf et al., eds., tenth edition, 1983, pp.1789-1801, McGraw-Hill, New York, incorporated herein by reference.
Current methods of identifying hepatitis B virus or its variants have depended on in vitro culture of the virus, radioimmunoassay, or genomic type identification after extraction of the viral DNA or RNA. However, for medical screening, diagnostic, or treatment purposes, these techniques do not always provide the necessary sensitivity. Dienstag, J.L., et al., in Harrison's Principles of Internal Medicine, R.G. Petersdorf et al., eds, tenth edition, 1983, pp.1789-1801. In addition, methods like radioimmunoassay may non-specifically detect the presence of viral antigens without providing information about the specific subtype.
The polymerase chain reaction (PCR) is a powerful technique for the amplification of specific DNA sequences. Cohen, S.N., U.S. Pat. No. 4,293,652; Erlich, H.A. et al., EP 258,017; Mullis, K.B., EP 201,184; Mullis et al., EP 200,362; Saiki, R.K., et al., Science 239:487-491 (1988); Mullis, K.B. et al., Meth. Enzymol. 155:335-350 (1987); Scharf, R.K., et al., Science 233:1076-1079 (1986) and Saiki, R.K., et al., Science 230:1350-1354 (1985).
The polymerase chain reaction technique has the ability to amplify a DNA sequence several orders of magnitude in a few hours, and has been used for the detection of low levels of viral sequences, Kwok, S. et al., J. Virol. 61:1690-1694 (1987), including hepatitis B, Kaneko, S., et al., Hepatology 8:1222 (1988); cloning of low-abundant DNA sequences, Lee, M.S., et al., Science 237:175-178 (1987); detection of malignant cells with chromosomal rearrangements, Lee, M.S., et al., Science 237:175-178 (1987); amplification of somatic mutational activation of cellular oncogenes in human tumors, Almoguera, C., et al., Cell 53:549-554 (1988); and in the analysis of clinical and forensic samples for the detection and identification of individual DNA genotype, Marx, J.L., Science 240:1408-1410 (1988), and haplotype, Li, H. et al., Nature 335:414-417 (1988). The use of the polymerase chain reaction as a DNA diagnostic technique has been recently reviewed, Landegren, U., et al., Science 242:229-237 (1988), incorporated herein by reference.
The polymerase chain reaction is based on the use of oligonucleotide primers complementary to sequences flanking a particular region of interest for primer-directed DNA synthesis in opposite and overlapping directions. With repeated cycles of high-temperature template denaturation, oligonucleotide primer reannealing, and polymerasemediated extension, DNA sequences can be faithfully amplified several hundred-thousand fold. The amplified sequences are remarkably accurate so one can reliably determine the nucleotide sequences immediately after the polymerase chain reaction.
In theory, only one copy of the target gene need be present in a sample for the polymerase chain reaction to adequately target and amplify it. For example, the polymerase chain reaction amplification technique has been used to analyze the DNA in an individual diploid cell and a single sperm. Li, H. et al. Science 335: 414-417 (1988). Ou, C.Y., et al., Science 239:295-297 (1988), has suggested the use of the polymerase chain reaction for the detection of HIV-1 virus in DNA from peripheral blood mononuclear cells.
However, use of the polymerase chain reaction is not immediately applicable to all samples. For example, it is not possible to directly test blood or serum using the polymerase chain reaction method because serum contains many inhibitors of the PCR technique. Studies utilizing the polymerase chain reaction to study blood cells have had to first isolate DNA from the cells by phenol or other similar, suitable techniques known in the art for isolation and concentration of DNA. This results in a large loss of sensitivity.
Thus, there remains a need for methodology, applicable to serum and other biological samples, for the rapid identification of low levels of pathological entities. Such would be methodology that does not require DNA isolation or prolonged incubation in vitro, that is sensitive enough to detect the presence of a extremely dilute levels of organisms and macromolecular entities in a sample and which promotes the cloning and genetic analysis of the pathological entity. Such methodology would still be technically simple enough to be embodied as a kit, and amenable for use as a routine screening method.